Project Summary/Abstract Fluorescence fluctuation spectroscopy (FFS) is an attractive technique for cellular applications. It determines kinetic and molecular properties of proteins with submicron resolution and single molecule sensitivity in the living cell. Brightness is a unique FFS parameter that specifies the average fluorescence intensity of a protein complex. Because brightness is directly proportional to the number of labeled proteins in the complex, it identifies the oligomeric state of the labeled protein. This conceptual idea has been successfully applied to measure the stoichiometry and binding curve of proteins in the living cell. We seek to build on this success by proposing projects with the potential for significant impact on quantitative cell studies that continue to push the envelope of the FFS method. The formation of ternary or higher-order protein complexes are ubiquitous features of protein interactions and play an important role in the regulation of cellular processes. However, current methods are mostly limited to the detection of interactions involving two proteins inside the cell. We seek to overcome this technical bottleneck and develop an FFS approach with the capacity to detect and characterize the interactions of ternary protein systems in the living cell. Another topic of interest is the nuclear envelope (NE), which consists of a double membrane layer that separates the nuclear and cytoplasmic compartments. It is now being recognized that the NE integrates a number of important cell functions, which include the regulation of signal transduction pathways and mechanical force transduction between the cytoplasm and the nucleus. The NE has over 60 distinct membrane proteins, whose physical and functional interactions remain largely unexplored. Characterization of these interactions will be crucial to understand the cell biology of the NE and to develop treatments for the growing range of human disorders linked to NE proteins. To facilitate such research, we propose to develop a quantitative FFS technique that explores the interaction and oligomerization of NE proteins inside the living cell. The environment of the NE poses a unique challenge for brightness measurements, which will be addressed by a novel analysis approach. We next propose to complement FFS with photoactivated localization microscopy (PALM) in order to harness the strength of both techniques. While PALM determines stoichiometry and surface density of membrane proteins at low concentrations, which are often closer to physiological conditions, FFS complements these results at high surface densities, which facilitates the detection of weak interactions. We will implement combined FFS and PALM studies and perform tests on simple model systems before moving to applications. These projects are expected to open up new avenues for exploring protein-protein interactions in cells with potential applications ranging from basic research in cell biology to pharmaceutical drug screening. Advances in FFS could help fight diseases by providing detailed information about protein interactions and may lead to the identification of targets for drug development.